Asunto(s)
Adhesión a Directriz , Guías de Práctica Clínica como Asunto , Pautas de la Práctica en Medicina , Trombocitemia Esencial/diagnóstico , Trombocitemia Esencial/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Sustitución de Aminoácidos , Examen de la Médula Ósea/estadística & datos numéricos , Colombia Británica , Estudios de Cohortes , Femenino , Hospitales Universitarios , Humanos , Janus Quinasa 2/genética , Masculino , Registros Médicos , Persona de Mediana Edad , Mutación , Inhibidores de Agregación Plaquetaria/uso terapéutico , Estudios Retrospectivos , Análisis de Supervivencia , Trombocitemia Esencial/genética , Organización Mundial de la SaludRESUMEN
Mammalian target of rapamycin complex 1 (mTORC1) phosphorylates proteins such as eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and the S6 kinases. These substrates contain short sequences, termed TOR signalling (TOS) motifs, which interact with the mTORC1 component raptor. Phosphorylation of 4E-BP1 requires an additional feature, termed the RAIP motif (Arg-Ala-Ile-Pro). We have analysed the interaction of 4E-BP1 with raptor and the amino acid residues required for functional RAIP and TOS motifs, as assessed by raptor binding and the phosphorylation of 4E-BP1 in human cells. Binding of 4E-BP1 to raptor strongly depends on an intact TOS motif, but the RAIP motif and additional C-terminal features of 4E-BP1 also contribute to this interaction. Mutational analysis of 4E-BP1 reveals that isoleucine is a key feature of the RAIP motif, that proline is also very important and that there is greater tolerance for substitution of the first two residues. Within the TOS motif, the first position (phenylalanine in the known motifs) is most critical, whereas a wider range of residues function in other positions (although an uncharged aliphatic residue is preferred at position three). These data provide important information on the structural requirements for efficient signalling downstream of mTORC1.
Asunto(s)
Proteínas Portadoras/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Arginina/metabolismo , Línea Celular , Factor 4E Eucariótico de Iniciación/genética , Glutatión Transferasa/metabolismo , Humanos , Isoleucina/metabolismo , Riñón/citología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Unión Proteica , Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Serina-Treonina Quinasas TOR , TransfecciónRESUMEN
PRAS40 binds to the mTORC1 (mammalian target of rapamycin complex 1) and is released in response to insulin. It has been suggested that this effect is due to 14-3-3 binding and leads to activation of mTORC1 signalling. In a similar manner to insulin, phorbol esters also activate mTORC1 signalling, in this case via PKC (protein kinase C) and ERK (extracellular-signal-regulated kinase). However, phorbol esters do not induce phosphorylation of PRAS40 at Thr(246), binding of 14-3-3 proteins to PRAS40 or its release from mTORC1. Mutation of Thr(246) to a serine residue permits phorbol esters to induce phosphorylation and binding to 14-3-3 proteins. Such phosphorylation is apparently mediated by RSKs (ribosomal S6 kinases), which lie downstream of ERK. However, although the PRAS40(T246S) mutant binds to 14-3-3 better than wild-type PRAS40, each inhibits mTORC1 signalling to a similar extent. Our results show that activation of mTORC1 signalling by phorbol esters does not require PRAS40 to be phosphorylated at Thr(246), bind to 14-3-3 or be released from mTORC1. It is conceivable that phorbol esters activate mTORC1 by a distinct mechanism not involving PRAS40. Indeed, our results suggest that PRAS40 may not actually be involved in controlling mTORC1, but rather be a downstream target of mTORC1 that is regulated in response only to specific stimuli, such as insulin.
Asunto(s)
Proteínas 14-3-3/metabolismo , Ésteres del Forbol/farmacología , Fosfoproteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular , Humanos , Insulina , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos , Fosforilación , Unión Proteica , Proteínas , Serina-Treonina Quinasas TORRESUMEN
Signaling through the mammalian target of rapamycin complex 1 (mTORC1) is positively regulated by amino acids and insulin. PRAS40 associates with mTORC1 (which contains raptor) but not mTORC2. PRAS40 interacts with raptor, and this requires an intact TOR-signaling (TOS) motif in PRAS40. Like TOS motif-containing proteins such as eIF4E-binding protein 1 (4E-BP1), PRAS40 is a substrate for phosphorylation by mTORC1. Consistent with this, starvation of cells of amino acids or treatment with rapamycin alters the phosphorylation of PRAS40. PRAS40 binds 14-3-3 proteins, and this requires both amino acids and insulin. Binding of PRAS40 to 14-3-3 proteins is inhibited by TSC1/2 (negative regulators of mTORC1) and stimulated by Rheb in a rapamycin-sensitive manner. This confirms that PRAS40 is a target for regulation by mTORC1. Small interfering RNA-mediated knockdown of PRAS40 impairs both the amino acid- and insulin-stimulated phosphorylation of 4E-BP1 and the phosphorylation of S6. However, this has no effect on the phosphorylation of Akt or TSC2 (an Akt substrate). These data place PRAS40 downstream of mTORC1 but upstream of its effectors, such as S6K1 and 4E-BP1.